This project has two primary aims. The first aim is to understand how proteins that are destined to travel through the secretory pathway are targeted to transport sites in the endoplasmic reticulum (ER) in eukaryotic cells or the cytoplasmic membrane (CM) in bacteria. For the last decade we have been investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its membrane-bound receptor in this process. Although SRP was initially believed to exist only in eukaryotic cells, the sequencing of a large number of microbial genomes in recent years has demonstrated that the particle is found in most (if not all) organisms. Previous studies have shown that in mammalian cells SRP recognizes the "signal sequences" found on virtually all secreted and membrane proteins as they emerge during translation and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. A few years ago we demonstrated that bacterial SRP has a somewhat more restricted function in that it only targets integral membrane proteins to the CM. Consistent with the work of other laboratories, we found that most secreted proteins, by contrast, are targeted to the CM by molecular chaperones. In recent studies we have continued to analyze the mechanism by which SRP recognizes the highly diverse family of signal sequences found on different proteins and then releases them at the surface of the ER (or bacterial CM) in a regulated fashion. By studying this problem we expect to obtain insight into the function and regulation of proteins that possess broad substrate specificity. In the past year we have also conducted a detailed investigation of the role of molecular chaperones in protein targeting in E. coli. In one study we unexpectedly found that an abundant bacterial-specific chaperone called trigger factor (TF) has a unique effect on protein export. Unlike most chaperones, which enhance or accelerate protein export, TF retards the secretion process. The simplest explanation of the data is that TF sequesters nascent polypeptides in a fashion that promotes the proper folding of cytoplasmic proteins, but that slows the export of presecretory proteins. In a second study we obtained evidence that DnaK, another important chaperone in E. coli, has a unique ability to maintain exported proteins in a loosely-folded, transport-competent state for a prolonged period of time. The second aim of the project is to elucidate the function of a universal protein conducting channel or "translocon" that is found in both the ER and the bacterial CM. We are particularly interested in understanding how the translocon catalyzes two related but seemingly distinct processes, namely the complete transfer of presecretory proteins across a membrane and the integration of membrane proteins into a lipid bilayer. In addition, we would like to determine why the core of the translocon is evolutionarily conserved, but the peripheral subunits are not. We expect that these experiments will provide significant insight into the function of membrane-bound channels.